Cyanide ion in production of vitamin b12



Patented Sept. 1, 1953 UNITED STATES oiFFIcE New- J ers'eyi Noprawing. @nationality-11';1950, serialNo. 161,484

10 Claims.- l (Cl. 1957-414) 634-5 that vitamin' B12 'canlbe obtained from ferL mentation media produced' by' the' propagation of anumber. of different microorganisms', and that the crystalline product that obtained is identical with the' crystallinev vitamin B12 pre viously obtained from liver. Although'the chemicall structure of vitamin B112 is not yet completely kn'c'wn; it'has been determined-that vitaminBiz `contains a characteristic CN group in the molecule.

Procedures for obtaining vitamin B12 from Streptomyces griseus fermentation liquors are fully disclosed and claimed in a pending appli-- cation by our colleagues Rickes and Wood, Sea rial No. 20,356, led' April'lO, 1948A (nowab'a'ndoned); and in a continuation-impart of said applica-tion, i. e., SerialN. 38,176; ledlJii-ly.v 10, 1948,110WU5 S. PatentN- 2,595,499, dat'edMay 6; 1952,' proceduresfare disclosed andclaim'edfor ber" of-v different organisms of* thel sub`phylum fungi. In these various fermentation processes, it has been found tnat'in'- addition" to'v vitamin-"B12, substances-are formed which have similarLLD-'and APF'activty but" Which'diffl from vitamin 1Biz' 'in eontainiirggno CNgrOupi These substances can be converted 'toY vitamin B12 by 'reaction with a'sub stance 'rurnisliing a source orey'anide 'ion `fas' more fully''di's'closeol':'and` 'claimed lin `an f application `by our' colleague' Frank J Vflff Serial No.` 120,009', 1d"OctOb`er`5f 1949," 110W Uf S.' Patent' NO. 2,530,416', dated" November' 21j. 1950'.' Tliepnverta'bili'ty'of 'these substances Y`to'vitamin llz'further demonstrates their very'l closel relationship to vitaminBiz, and"w'e-willlrefrto-theseiclosely related "substancesfhereinarter las 'vitamin' Bia-like materialdior' vitamin Biz-like substances. There isb aratii'erf strong indication, however; that the so-called vitamin' B12-like materials 'may consist pred'crrninantly of`- a: derivative of. vitamin B12 characterized byth'e"y absence of the CN group inf th'eimolecule. Such" a derivative has been prepared fby hydrogenation:ofivitamin B12 `in the presencev of platinum" oxide' catalyst. In this connection, attentionlisfdirected`to a' publication by; Kaczkaletiali, J; A'.C.'S. 71, 1514, reporting this" catalytic'freduction" of vitamin B12' and identifyingthe product 'as :vitaminv Bies.

We-have" now discovere'dtliat' a' marked increase' in` yield." of? vitamin B12 and of vitamin Bizelikef materialsV (estimated as vitamin' Biza) can be'obtainedlby 'conducting fermentation with various vita'm'in' Bia-producing organisms inthe presence'o'i ais'mallamountof a substance from wliihtheorganismv may obtain the' CN group. As vvn'i'orefilly hereinafter described; the fermentation canbei conducted in' thefpresen'ce of a substan'cei'w'hichlfurnlshes'to the organism a directsour'ce'i of cyanide ion r such as 'a simple cyanidewhieli'ionizes'in'solution, or in the presence of'a compleXiromwhich the 'organism can liberate the cyanide groupin utilizable fform'.'

There have' been? various disclosures in the prionarti'showingtheiuse' of cyanides as inhibitorsV of enzyme? systemsiin" microorganisms, the eiect 'of "such inhibition of 'enzyme-'systems being to' alter 'the'na'tur'efand amounts' of fermentation products obtained: In' ourimproved process `for producing' vitamin-B12 land vitamin' B12-like materials byferm'entation', however, experimental results are believedI to' indicate' that the in-' crease'd'yieldsfare"due not to'any inhibition of the organism or its 'associated enzyme system, but rather 'to a specifcprecursor action provided by thev presence ofV cyanide ion available to the organism in lthe medium'.

While' We do -not'wish to' b'e'bound 'by' a theoretical explanation' ofthi-s'phen'omenon, the fol'- lowin'g observations are Vbelieved to'be 'signicant lThe vitamin'lBi'z-likef materials which are'pro'- duced --along'w'itli vitamin B12 :during fermentation' are .very'sir'nilarr tovitamin :B12 Ibut do .not contain the=cyanidegroup-- inasmuch as fer-'- mentati-ngl brothsl When producing vitamin B12 arefoftentmildlyreducing; it would appear that the vitamin B12-like materials are formed at least in part in the medium by this reducing action on the vitamin B12 actually synthesized by the organism. While the cyanide ion could function, and possibly does function in part, to convert the vitamin B12-like materials to vitamin B12 (by substitution of the cyanide ion in the molecule as more fully disclosed and claimed in the above-mentioned application of our colleague Frank J. Wolf, Serial No. 120,009, filed October 6, 1949), the experimental evidence also clearly demonstrates that the increase in vitamin B12 activity which we obtain is markedly greater than the increase which can be accounted for by such conversion of vitamin B12-like materials to vitamin B12. In other words, the total vitamin B12 activity, i. e., vitamin B12 plus vitamin B12- like materials is markedly increased by our improved process, thus demonstrating that the cyanide ion has a definite precursor action stimulating synthesis of vitamin B12 by the organism during fermentation.

The apparent action of cyanide ion as a pre- Y cursoi' in our improved process is a factor which is both surprising and unexpected in View of prior knowledge concerning use of cyanide ion in fermentation processes. In this connection, there is a distinct difference between the prior art procedues of affecting an enzyme system by the presence of cyanide so as to cause an increase in yield of some fermentation product which contains no cyanide group, and our improved process wherein the cyanide ion supplied in the medium is actually utilized by the organism in the synthesis of Vitamin B12.

In carrying out improved fermentation procedures in accordance with the present invention, fermentation media otherwise capable of fostering production of vitamin B12 and vitamin B12-like materials are supplemented by addition of a substance which will furnish to the organism a source of cyanide ion. By this is meant not only compounds which ionize in the medium to produce free cyanide ions, but also certain complexes containing the cyanide group in a form which appears to be acted upon by the organism to liberate cyanide ion. Thus, for example, ionizable cyanides such as hydrogen cyanide, ammonium cyanide, alkali metal cyanides and alkaline earth metal cyanides can readily be employed in our process, but in addition it has been found that certain complexes such as alkali metal ferrocyanides and ferricyanides effect a similar precursor action apparently due to liberation of cyanide ion by the microorganism.

The amount of the substance furnishing a source of cyanide ion which is required to stimulate production of vitamin B12 and vitamin B12- like materials is extremely small, as little as 0.1 p. p. rn. of cyanide ion showing a significant increase. Larger amounts of the substance furnishing cyanide ion can be employed, including amounts suflicient to furnish about 50-100 p. p. m. of cyanide ion. Further increases should generally be avoided, however, since free cyanide ion in excess of about 100 p. p. m. appears to have a toxic effect on the various vitamin B12-producing organisms, resulting in a retarding of the growth during fermentation and falling off of the Vitamin B12 production. Complexes such as alkali metal ferrocyanides and ferricyanides do not furnish free cyanide ion in solution, and can be employed in larger amounts without danger of retarding growth during fermentation. For best results, it would appear that the substance 4 furnishing a source of cyanide ion should be present in the fermentation medium in an amount to make available to the organism about 1-10 p. p. m. of cyanide ion.

The substance furnishing a source of cyanide ion is preferably incorporated in the medium at the start of the fermentation, the entire amount being added before fermentation is started, or separate portions of the total amount being added at the start of the fermentation and at intervals as the fermentation progresses. While an increased yield of vitamin B12 and vitamin B12-like materials is also obtained when a substance furnishing a source of cyanide ions is added after 'fermentation has been in progress for a period of time, it is generally found that in such instances the amount of increase is not as great as when cyanide ion is present from the start of the fermentation.

The following examples show how improved fermentation procedures in accordance with the present invention can be carried out, but it is to be understood that these examples are given by way of illustration and not of limitation.

Example 1 In 250 ml. Erlenmeyer flasks were placed 40 ml. quantities of a medium containing 1% of an enzymatic digest of casein. 0.3% beef extract, 0.5% sodium chloride, 10 p. p. m. cobalt nitrate hexahydrate and distilled water, at pH 7.0. To the flasks were added either 0, 0.1, 1, '10, 100, 1000 or 5000 p. p. m. of cyanide ion supplied as potassium cyanide. The media were then sterilized by autoclaving at 121. C. for 15 minutes.

To each flask was then added 1 ml. of a 40 hour vegetative culture of a grisein-producing strain of S. grz'scus, grown in a medium containing the same ingredients as those described above. The flasks were then plugged with cotton, placed on a 11/2 amplitude shaker, and shaken for hours at 28 C. Samples were taken from the flasks, examined for growth, tested for pH, and assayed against L. lac-tis Dorner. The following results were obtained:

Average p. p. m. CN- Growth pH S1133? u./1nl.

Quantitative evaluations of vitamin B12 and of vitamin B12-like materials, measured as vitamin B122. were made by paper strip chromatographic technique. A control culture and a culture containing 1 p. p. m. of cyanide ion were evaluated as follows:

Cultures were acidified to pH 2.5, filtered, and neutralized. Ten ml. of filtrate was saturated with cresol and extracted with 2.5 mi. of carbon tetrachloride-cresol (3-1) (this extraction procedure is fully disclosed and claimed in an application of Denkewalter, Malcolmson and Hughey, Serial No. 179,888, filed August 16, 1950). Paper strips impregnated with KH2PO4 were spotted With 0.1 ml. quantities of extract and developed three days with water-saturated nbutanol. (By this technique, it has been found that pure vitamin B121. and vitamin B12 localize in spots at about 2 inches and '1 inches respectity of vitamin B12 contained in the culture Example In 250 ml. Erlenmeyer flasks were placed 50 ml. quantities of a medium containing 2% of an enzymatic digest of casein 0.5% beef extract, 0.25% K2HPO4 and 10 p. p. m. cobalt nitrate hexahydrate. All flasks were sterilized by autoclaving at 120 C. for 20 minutes. A water solution of KCN was sterilized by filtration through an ultra-fine sintered glass filter and added to each flask aseptically as indicated. 'All asks were inoculated with 1 ml. of a 24 hr. submerged culture of an isolate' from cow rumen contents identified as an Alkaligenes faecalis, grown in a medium containing the same ingredients as those described above. The cultures were incubated at 28 C. on a 11/ amplitude shaker for three days. The harvested cultures were autoclaved 15 minutes at 120 C. to liberate the total LLD active substances, and assayed for LLD activity by the cup assay procedure with L. Zactis Dorner as a test organism and vitamin B12 as a standard. Samples were centrifuged to remove cellular debris and m1. of the supernatant extracted with 2.5 ml. of cresol-carbon tetrachloride, as described in Example l. The solvent layer was separated and 0.04 ml. spotted on a paper strip impregnated with KH2PO4 and developed 3 days with water-saturated n-butanol. The LLD active area characteristic of vitamin B12 was located and leached with water for quantitative assay. Alkaligenes faecalis is characterizde by production of a large amount of LLD active substances which do not appear to be chemically similar to vitamin B12. In the presence of potassium cyanide, vitamin B12 was produced, as shown by paper strip chromatography.

Amount of KON added L. lactic asalyulrents per ml.

Beffgoc' Atecf ofs LLD activity 210111215 1331511 u a o Lu u o p' p m p. p m. of culture paperzlslrp Bu Vitamin B12 contains 11,000 ILD units per microgram. Therefore, in the above experiment, from cultures grown on medium supplemented with 8 p. p. m. KCN before inoculation, 0.079',l of vitamin B12 per ml. of culture was separated, and from cultures having an additional 8 p. p. m. KCN added at the 24th hour of incubation, 0.1643/ of vitamin B12 per ml. of culture was separated.

Example 6 A medium containing 4% distillers dried solubles and 10 p. p. m. Co(NO3)2.6H2O in Water was prepared and distributed in 40 cc. quantities in 250 cc. flasks. Suicient potassium cyanide was added to the asks to furnish 0.1, 1.0 and l0 p. p. m. of CN ion in the medium. Flasks were plugged with cotton and sterilized by autoclaving at 121 C. for 15 minutes.

Vegetative inoculum was prepared by culturing in a beef extract-enzymatic digest of casein type medium an unidentied microorganism belonging to the genus Streptomyces, isolated from a sample of decomposed barnyard litter. This culture was found to be an exceptionally good culture for production of vitamin B12.

The media were inoculated with one m1. of the above vegetative inoculum and incubated for four days at 28 C. on a shaker.

At the completion of the fermentation, the total LLD activity of the culture Was determined by L. lactis titrimetric assay. The vitamin B12 and B12-like material was extracted and quantitatively measured by the paper strip chromatographic technique described in Example l. Results are recorded in the following table.

L. laciis assay, units per ml.

of culture Medium supplement B12 and B11- LLD actwlty like material of culture units Various changes and modifications in the foregoing procedure will occur to those versed in the art, and to the extent that such changes and modifications fall within the purview of the appended claims, it will be understood that they constitute part of our invention.

We claim:

l. In the production of vitamin B12 and vitamin B12-like materials by fermentation of a nutrient medium with a lvitamin B12 producing organism, the improvement which comprises conducting the fermentation in the presence cf a substance furnishing a source of cyanide ion in an amount within the range of about 0.1 to 100 parts per million.

2. The process as defined in claim l wherein the fermentation is conducted in the presence of a substance furnishing a source of cyanide ion in an amount Within the range of about 1 to 10 parts per million.

3. In the production of vitamin B12 and vitamin B12-like materials by fermentation of a nutrient medium with a vitamin B12 producing organism, the improvement which comprises conducting the fermentation in the presence of an ionizable cyanide in an amount to provide a cyanide ion concentration Within the range of about 0.1 to 100 parts per million.

4. The process as dened in claim 3 wherein the ionizable cyanide is an alkali metal cyanide.

5. The process as defined in claim 3 wherein the ionizable cyanide is potasisum cyanide.

6. The process as dened in claim 3 wherein the ionizable cyanide is present in an amount to provide a cyanide ion concentration within the vmedium with a Vitamin B12 producing organism,

vthe improvement which comprises conducting the fermentation in the presence of an alkali metal ferrocyanide furnishing a source of cyanide ion in an amount Within the range of about 0.1 to 100 parts per million.

8. In the production of vitamin B12 and vitamin B12-like materials by fermentation of a nutrient medium with a vitamin B12 producing organism, the improvement which comprises conducting the fermentation in the presence of an alkali metal ferricyanide furnishing a source of cyanide ion in an amount Within the range of about 0.1 to 100 parts per million.

9. In the production of vitamin B12 and Vitamin B12-like materials by fermentation of a nutrient medium with a vitamin B12 producing organism, the improvement which comprises conducting the fermentation in the presence of a substance furnishing a source of cyanide ion in an amount within the range of about 0.1 to 100 parts per million, said substance being added to the medium at the start of fermentation.

10. In the production of vitamin B12 and vitamin B12-like materials by fermentation of a nutrient medium with a vitamin B12 producing organism, the improvement which comprises conducting the fermentation in the presence of a 10 substance furnishing a source of cyanide ion in an amount within the range of about 0.1 to parts per million, said substance being added to the medium at the start of the fermentation and again at periodic intervals as the fermentation progresses.

LLOYD E. MCDANIEL. HAROLD B. VOODRUFF.

References Cited in the file of this patent UNITED STATES PATENTS Name Date Wallerstein June 10, 1930 Wolf Nov. 21, 1950 OTHER REFERENCES Number 

1. IN THE PRODUCTION OF VITAMIN B12 AND VITAMIN B12-LIKE MATERIALS BY FERMENTATION OF A NUTRIENT MEDIUM WITH A VITAMIN B12 PRODUCING ORGANISM, THE IMPROVEMENT WHICH COMPRISES CONDUCTING THE FERMENTATION IN THE PRESENCE OF A SUBSTANCE FURNISHING A SOURCE OF CYANIDE ION IN AN AMOUNT WITHIN THE RANGE OF ABOUT 0.1 TO 100 PARTS PER MILLION. 